Human Umbilical Cord-Derived Mesenchymal Stem Cells Do Not Undergo Malignant Transformation during Long-Term Culturing in Serum-Free Medium

Background: Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However, the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. Methods: hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st, 3rd, 5th, 8th, 10th, and 15th passage, the differentiation of MSCs into osteogenic, chondrogenic, and adipogenic cells was detected, and MTT, surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0, 8, and 15. The telomerase activity of hUC-MSCs at passage 1-15 was analyzed. Results: Flow cytometry analysis showed that very high expression was detected for CD105, CD73, and CD90 and very low expression for CD45, CD34, CD14, CD79a, and HLA-DR. MSCs could differentiate into osteocytes, chondrocytes, and adipocytes in vitro. There was no obvious chromosome elimination, displacement, or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further, no tumors formed in rats injected with hUC-MSCs (P-15) cultured in serum-free and in serum-containing conditions. Conclusion: Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P-15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium, these cells are suitable for cell therapy.